US20140020454A1 - Liquid chromatography column, and method for analyzing hemoglobin - Google Patents

Liquid chromatography column, and method for analyzing hemoglobin Download PDF

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Publication number
US20140020454A1
US20140020454A1 US14/005,351 US201214005351A US2014020454A1 US 20140020454 A1 US20140020454 A1 US 20140020454A1 US 201214005351 A US201214005351 A US 201214005351A US 2014020454 A1 US2014020454 A1 US 2014020454A1
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United States
Prior art keywords
column
hemoglobin
liquid chromatography
present
filler particles
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Abandoned
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US14/005,351
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English (en)
Inventor
Takuya Yotani
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Sekisui Medical Co Ltd
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Sekisui Medical Co Ltd
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Assigned to SEKISUI MEDICAL CO., LTD. reassignment SEKISUI MEDICAL CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: YOTANI, TAKUYA
Publication of US20140020454A1 publication Critical patent/US20140020454A1/en
Abandoned legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G01N30/6052—Construction of the column body
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8822—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G01N30/6004—Construction of the column end pieces

Definitions

  • the present invention relates to a column for liquid chromatography.
  • the present invention also relates to a method for analyzing hemoglobin using the column for liquid chromatography.
  • hemoglobin A1c is glycated hemoglobin that has blood sugar chemically bound to an N-terminal of a ⁇ -globin chain of hemoglobin.
  • a proportion of hemoglobin A1c in the hemoglobin i.e., a ratio of glycated hemoglobin to the total of glycated hemoglobin and non-glycated hemoglobin, is considered to reflect an average blood glucose level in a period of one to two months.
  • a hemoglobin A1c value (%) representing the proportion of hemoglobin A1c in the total hemoglobin does not exhibit temporal fluctuations and thus has been widely used as an indicator for diabetic diagnosis.
  • Patent Literature 2 discloses a column produced using engineering plastics having excellent strength (pressure resistance) as typified by polyetheretherketone (PEEK). The use of such a column can suppress non-specific adsorption of components derived from living organisms.
  • PEEK polyetheretherketone
  • the present invention relates to a column for liquid chromatography including a column pipe and an end fitting, wherein the column pipe and the end fitting comprise polyethylene or polymethylmethacrylate.
  • the use of highly hydrophobic materials for the column may be considered.
  • highly hydrophobic materials specifically, materials with a water contact angle of 90° or more
  • separation performance is reduced, depending on the type of materials.
  • some columns formed using relatively low hydrophobic materials can successfully detect target objects when these columns are used for measuring samples such as hemoglobin. Therefore, the degree of hydrophobicity of materials for columns is not considered to have an inevitable effect on separation performance of hemoglobin and the like.
  • the present inventors found that the use of polyethylene or polymethylmethacrylate as materials of a column pipe and an end fitting enables low-cost production of a column for liquid chromatography that allows suppression of non-specific adsorption of components derived from living organisms and ensures accurate measurement.
  • the present invention is accomplished based on the above finding.
  • the column for liquid chromatography of the present invention (hereinafter also referred to as the “column of the present invention”) includes a column pipe and an end fitting each formed from polyethylene or polymethylmethacrylate. Non-specific adsorption of components derived from living organisms can be suppressed and an accurate measurement can be achieved not only when highly hydrophobic polyethylene is used but also when relatively low hydrophobic polymethylmethacrylate is used. Further, because polyethylene and polymethylmethacrylate are commodity resin, the column of the present invention can be produced at low cost and can be incinerated, making it easily disposable.
  • the column of the present invention can be produced by, for example, injection molding, extrusion molding, cutting, or the like.
  • the shapes of the column pipe and the end fitting are not particularly limited.
  • the column pipe and the end fitting are connected to each other by a ferrule or a screw, for example.
  • an eluent and a sample are introduced into the column under specific conditions, and the sample is thereby measured.
  • the use of a column having a column pipe and an end fitting formed from polypropylene, polyethylene terephthalate, polycarbonate, or the like tends to cause non-specific adsorption to the inner surface of the column; however, the use of the column of the present invention allows suppression of non-specific adsorption to the inner surface of the column, and thus hemoglobin can be analyzed with high separation performance.
  • a liquid chromatograph used for the method for analyzing hemoglobin of the present invention has a structure in which the column of the present invention packed with filler particles is connected to a known liquid chromatograph equipped with a pump for delivering an eluent, a detector, and the like.
  • the flow pressure of the column during analysis is determined by the particle size and particle size distribution of filler particles, column inside diameter, column length, eluent flow rate, and the like. Because the flow pressure varies depending on the conditions, it may be suitably adjusted within a range that results in desired separation performance. To allow analysis of hemoglobin with high separation performance without causing problems such as damage to the column and leakage from the column, the flow pressure of the column during analysis is preferably 4.0 MPa or less, and more preferably 3.0 MPa or less.
  • the flow pressure of the column is measured by reading a pressure value from a pressure gauge connected between the delivery pump and the column.
  • the average particle size of filler particles, inside diameter of the column, column length, and eluent flow rate, which also significantly affect separation performance, are preferably adjusted within the following range.
  • the average particle size of filler particles preferably has a lower limit of 2 ⁇ m and an upper limit of 20 ⁇ m. In the case where the average particle size of filler particles is less than 2 ⁇ m, it increases the flow pressure of the column and thus may cause problems such as damage to the column and leakage from the column. Filler particles with an average particle size of more than 20 ⁇ m result in a decrease in separation performance. When such filler particles are used in measurement of hemoglobin A1c, separation of hemoglobin may be insufficient.
  • a more preferred lower limit of the average particle size of filler particles is 5 ⁇ m, and a more preferred upper limit thereof is 15 ⁇ m.
  • the average particle size of filler particles is a value measured using a particle size distribution measuring device (available from Particle Sizing Systems; Accusizer 780).
  • the inside diameter of the column of the present invention preferably has a lower limit of 2.0 mm and an upper limit of 6.0 mm.
  • a column having an inside diameter of less than 2.0 mm the linear velocity of the mobile phase flowing through the column is too high, and the flow pressure of the column is thus too high, which may cause problems such as damage to the column and leakage from the column.
  • a column having an inside diameter of more than 6.0 mm the analyte and mobile phase in the column are too dispersed, which may result in a decrease in separation performance.
  • a more preferred lower limit of the inside diameter of the column is 3.0 mm, and a more preferred upper limit thereof is 5.0 mm.
  • the length of the column of the present invention preferably has a lower limit of 10 mm and an upper limit of 50 mm.
  • a column with a length of less than 10 mm may result in a decrease in separation performance along with a decrease in the number of theoretical plates.
  • a column with a length of more than 50 mm results in an increase in the flow pressure of the column, which may cause problems such as damage to the column and leakage from the column.
  • a more preferred lower limit of the column length is 15 mm, and a more preferred upper limit thereof is 40 mm.
  • the eluent flow rate of the delivery pump preferably has a lower limit of 0.5 mL/min and an upper limit of 2.5 mL/min.
  • An eluent flow rate of less than 0.5 mL/min results in a long measurement time due to a slow flow rate.
  • An eluent flow rate of more than 2.5 mL/min results in an increase in the flow pressure of the column, which may cause problems such as damage to the column and leakage from the column.
  • a more preferred lower limit of the eluent flow rate is 1.0 mL/min, and a more preferred upper limit thereof is 2.0 mL/min.
  • Filler particles to be packed into the column of the present invention are those used in the conventionally well-known ion-exchange liquid chromatography. Filler particles including ion exchange groups and having the outermost surface hydrophilically treated with ozone water, such as those disclosed in JP Patent No. 3990713, are particularly suitable. The use of such filler particles allows measurement of samples such as hemoglobin with high separation performance even when the flow pressure of the column is low.
  • the column of the present invention preferably includes filters, each disposed upstream and downstream of the column for preventing leakage of filler particles in the column from a tubular container.
  • the filter is not for trapping contaminants, and it is sufficient if the filter can prevent leakage of filler particles from a tubular container.
  • a filter formed of paper, resin, metal, or the like may be used, for example.
  • a stainless steel filter having three layers with different pore sizes such as the one disclosed in JP-A 2006-189427, is particularly suitable.
  • the filtration surface of the filter may be formed in a circular shape or another shape.
  • the column preferably includes a pre-filter disposed upstream of the column for trapping contaminants.
  • the pre-filter may be arranged separately from the column of the present invention, or integrally arranged with the column in a single tubular container. In either case, the pre-filter is connected to a pipe such that the pre-filter is arranged upstream of the column.
  • an eluent used in the liquid chromatography is preferably a buffer containing a known salt compound or an organic solvent.
  • a buffer containing a known salt compound or an organic solvent include organic acids, inorganic acids, salt thereof, amino acids, and Good's buffers.
  • organic acids examples include citric acid, succinic acid, tartaric acid, and malic acid.
  • inorganic acids examples include hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boric acid, and acetic acid.
  • amino acids examples include glycine, taurine, and arginine.
  • the buffer may contain other common additives.
  • surfactants various polymers, hydrophilic low-molecular compounds, chaotropic ions, and the like may be suitably added to the buffer.
  • the salt concentration of the buffer preferably has a lower limit of 10 mmol/L and an upper limit of 1000 mmol/L. No ion-exchange reaction occurs when the buffer has a salt concentration of less than 10 mmol/L, and hemoglobin thus cannot be separated in some cases.
  • a buffer having a salt concentration of more than 1000 mmol/L may cause salt deposition, adversely affecting the system.
  • the method for analyzing hemoglobin of the present invention allows measurement of hemoglobin A0, hemoglobin A1c, hemoglobin F (fetal hemoglobin), hemoglobin A2, and the like. Abnormal hemoglobin variants such as hemoglobin S, hemoglobin C, hemoglobin D, and hemoglobin E can also be measured.
  • the present invention provides a column for liquid chromatography that can be produced at low cost and allows suppression of non-specific adsorption of components derived from living organisms.
  • surface treatment such as silicone resin coating must be applied to the column.
  • the column of the present invention does not require such surface treatment.
  • polyethylene and polymethylmethacrylate are commodity resins, they are inexpensive, which provides a significant economic advantage. Further, because the column of the present invention can be incinerated, it can be easily disposed of.
  • the present invention also provides a method for analyzing hemoglobin using the column for liquid chromatography.
  • the method for analyzing hemoglobin of the present invention allows suppression of non-specific adsorption of components derived from living organisms and ensures accurate measurement.
  • FIG. 1 is a chromatogram obtained by measuring hemoglobin using a polyethylene column.
  • FIG. 2 is a chromatogram obtained by measuring hemoglobin using a polymethylmethacrylate column.
  • FIG. 3 is a chromatogram obtained by measuring hemoglobin using a stainless steel column coated with silicone resin.
  • FIG. 4 is a chromatogram obtained by measuring hemoglobin using a non-surface-treated stainless steel column.
  • FIG. 5 is a chromatogram obtained by measuring hemoglobin using a polypropylene column.
  • FIG. 6 is a chromatogram obtained by measuring hemoglobin using a polyethylene terephthalate column.
  • FIG. 7 is a chromatogram obtained by measuring hemoglobin using a polycarbonate column.
  • FIG. 8 is a graph showing a relationship between columns of various materials in examples and comparative examples and half-widths of hemoglobin A1c peaks.
  • FIG. 9 is a graph showing a relationship between columns of various materials in examples and comparative examples and total peak areas.
  • a stainless steel (SUS316) column of Comparative Example 1 was coated with silicone resin using SR2410 (available from Dow Corning Toray Co., Ltd.).
  • the obtained filler particles had an average particle size of 10 ⁇ m and a CV value of 14% as measured using a particle size distribution measurement device (available from Particle Sizing Systems; Accusizer 780).
  • the obtained filler particles (0.5 g) were dispersed in 20 mL of 40 mmol/L phosphate buffer (pH 5.3) and stirred to form a slurry. After ultrasonic treatment for five minutes, the entire slurry was poured into a packing device (available from AS ONE Corporation) connected to each empty column (inside diameter of 4.6 mm ⁇ length of 15 mm). A delivery pump (available from GL Sciences; PU-614) was connected to the packing device, and the eluent was delivered at a pressure of 8 MPa to pack the column at constant pressure.
  • Hemoglobin A1c control analyte (available from Sysmex Corporation) was measured using the above-prepared columns. Table 2 shows measurement conditions. FIGS. 1 to 7 show chromatograms obtained from measurements using the columns of various materials.
  • FIG. 8 shows a relationship between the columns of various materials in examples and comparative examples and half-widths of hemoglobin A1c peaks.
  • SUS316 treated was used as the stainless steel column coated with silicone resin
  • SUS316 non-treated was used as the non-surface-treated stainless steel column.
  • FIG. 9 shows a relationship between the columns of various materials and total peak areas.
  • the total peak areas obtained with the PE column and the PMMA column were substantially the same as that obtained with the stainless steel column coated with silicone resin. However, the total peak areas obtained with other columns were clearly low.
  • the present invention provides a column for liquid chromatography that can be produced at low cost and allows suppression of non-specific adsorption of components derived from living organisms.
  • the present invention also provides a method for analyzing hemoglobin using the column for liquid chromatography.

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
US14/005,351 2011-03-22 2012-03-21 Liquid chromatography column, and method for analyzing hemoglobin Abandoned US20140020454A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2011-062936 2011-03-22
JP2011062936 2011-03-22
PCT/JP2012/057135 WO2012128276A1 (ja) 2011-03-22 2012-03-21 液体クロマトグラフィー用カラム及びヘモグロビン類の分析方法

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US (1) US20140020454A1 (ja)
EP (1) EP2690435B1 (ja)
JP (2) JP6109733B2 (ja)
CN (1) CN103443622B (ja)
WO (1) WO2012128276A1 (ja)

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JP2016027326A (ja) * 2014-06-26 2016-02-18 アークレイ株式会社 測定方法、測定装置および溶離液
CN113009025B (zh) * 2018-12-29 2023-04-07 江山德瑞医疗科技有限公司 一种测定值不受样本保存时间的影响糖化血红蛋白测定方法

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JPWO2012128276A1 (ja) 2014-07-24
CN103443622B (zh) 2015-10-14
JP2017102130A (ja) 2017-06-08
EP2690435B1 (en) 2022-10-05
CN103443622A (zh) 2013-12-11
EP2690435A4 (en) 2014-09-24
JP6109733B2 (ja) 2017-04-05
EP2690435A1 (en) 2014-01-29
WO2012128276A1 (ja) 2012-09-27

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